Effect And Mechanism Of Del-1 On Epidermal Neutrophil Infiltration In Psoriasis

Background:Psoriasis is a chronic recurrent inflammatory skin disease that is influenced by genetic and various environmental factors.The pathological features of psoriasis are abnormal proliferation and differentiation of keratinocytes 、 neutrophils infiltration into the epidermis forming microabscesses and T cell-dominant immune cell infiltration in the dermis.The pathogenesis of psoriasis is complex,and although it is still controversial,it is generally believed that the complex interaction between activated keratinocytes and infiltrating immune cells leads to the development of the disease.In recent years,the role of neutrophils in the pathogenesis of psoriasis has attracted more and more attention,and more and more evidences show that neutrophils play a crucial role in psoriasis.Neutrophils are one of the most important cell types in the skin of psoriatic lesions.They infiltrate into the dermis at the early stage and then enter the epidermis at the chronic stage,then neutrophils adhere to keratinocytes and accumulate in the epidermis to form microabscesses,which is one of the typical pathological manifestations of psoriasis.Due to the short half-life of neutrophils,they need to be continuously recruited to maintain the chronicity of psoriatic lesions.Studies have shown that anti-neutrophil therapy can not only alleviate imiquimod-induced psoriatic dermatitis in mice,but also has a good effect on psoriasis patients.Del-1 is an endogenous anti-inflammatory factor,and its genetically deficient mice showed significant neutrophil infiltration and elevated IL-17.The function of Del-1 depends on the "cell type" of origin.Endothelial cell-derived Del-1 blocks the interaction between neutrophils and vascular endothelial cells by antagonizing LFA-1 integrin on neutrophils,thereby inhibiting the adhesion of neutrophils to endothelium and subsequent transendothelial migration.Macrophage-derived DEL-1 can promote the clearance of apoptotic neutrophils,thereby promoting inflammation resolution.Whether human keratinocytes express Del-1 and its specific role remain unclear.Antagonizing neutrophil infiltration into the epidermis is a promising strategy for the treatment of psoriasis.Del-1,as a regulator of neutrophils,may alleviate the development of psoriasis by antagonizing the infiltration of neutrophils into the epidermis,which deserves further study.Research Objectives:The purpose of this study was to investigate the effect of keratinocyte-derived Del-1 on neutrophil infiltration into epidermis in psoriasis,and to explore the underlying molecular mechanism of its effect on neutrophil chemotaxis and adhesion function,providing theoretical basis for excessive neutrophil infiltration in the epidermis of psoriasis,and providing a new potential target for the treatment of psoriasis.Research Methods:1.Immunohistochemistry was used to locate and semi-quantify the expression of Del-1 in the epidermis of patients with psoriasis vulgaris,and RT-q PCR was used to detect the expression of Del-1 in the epidermis of patients with psoriasis by separating the epidermis of diseased skin and healthy skin.The expression of Del-1 in the skin lesions of psoriasis vulgaris patients and its correlation with IL-17 A were analyzed using GEO database data,and verified by RT-q PCR in the skin lesions of patients.RT-q PCR and Western Blot were used to detect the expression of Del-1 in psoriasis cell model.2.Ha Ca T cell lines with stable overexpression and knockdown of Del-1were constructed by using overexpression lentivirus and sh RNA lentivirus.Neutrophils were isolated from peripheral blood of healthy subjects and labeled with celltracker red fluorescent dye.Ha Ca T cells were stimulated with TNF-α to simulate psoriasis cell model.The experiment was divided into four groups: normal Ha Ca T cell(Ctrl group)was used as negative control,TNF-α-stimulated-Ha Ca T simulating psoriasis cell model(Ctrl+TNF-α group)as positive control,TNF-α-stimulated-overexpression-Del-1 Ha Ca T cell(OE-Del-1+TNF-α group)and TNF-α-stimulated-knockdown-Del-1 Ha Ca T cell as experimental groups.Cell culture supernatants of different groups were collected and placed in the lower pore plate of the Transwell chambers.The neutrophils suspension traced by red fluorescence were placed in the upper chamber of the Transwell device.The transwell device was incubated in the cell incubator for 1 h.Then the upper chamber was removed and the lower orifice plate of the Transwell chamber was photographed under a fluorescence microscope.Those with red fluorescence were the neutrophils that migrate to the lower layer.At the same time,neutrophils in the lower orifice plate of different groups were collected for counting,and the difference in the number of chemotactic neutrophils between different groups was analyzed.3.The cells of different groups(Ctrl,Ctrl+TNF-α,OE-Del-1 +TNF-α and sh-Del-1 +TNF-α groups)were inoculated in equal numbers in 96-well plates.When the degree of cell fusion was 100%,the red fluorescence labeled neutrophils suspension were gently added to the monolayer cells of each group and incubated in a cell incubator for 40 minutes.Then take out the well plate,discard the medium,and wash gently with PBS for 3 times to remove non-adherent neutrophils.Then fluorescence photos were taken,and 5 visual fields were taken from each hole.Image J was used to count the number of neutrophils with red fluorescence,and SPSS software was used for statistical analysis.4.The differential genes in the above four groups were screened by inflammatory response and autoimmunity PCR array plate and verified by RT-q PCR.The expression of adhesion molecule ICAM-1 was detected by RT-q PCR and Western Blot.5.The total protein and nuclear protein of the above four groups were extracted,and the phosphorylation and nuclear translocation of NF-κB-p65 in the four groups were detected by Western Blot to explore the molecular mechanism of Del-1 regulating inflammatory factors.Research Results:1.Immunohistochemical results of paraffin Del-1 in the skin of patients with psoriasis vulgaris and healthy persons suggest that Del-1 is constitutively expressed in keratinocytes of human skin,and the expression of Del-1 is decreased in the epidermis of patients.Normal tissues around the skin lesions that were pathologically diagnosed as nevus,seborrheic keratosis and other diseases were collected as healthy controls,and frozen tissues that were pathologically diagnosed as psoriasis vulgaris were collected as experimental groups.The epidermis was separated and RNA was extracted for RT-q PCR.The results showed that the expression level of Del-1 m RNA in the epidermis of patients was also reduced.Sequencing results of GEO database GSE13355 data set showed that del-1 expression was decreased in the skin lesions of psoriasis patients compared with healthy controls,and RT-q PCR verification was consistent with the database results.The data set GSE30999 with the largest number of patients was screened from GEO database,and the m RNA expression levels of DEL-1(EDIL3)and IL-17 A were extracted for correlation analysis.The results indicated that del-1 was negatively correlated with IL-17A(R =-0.45).In the psoriasis cell model simulated by Ha Ca T cells stimulated by TNF-α,the expression of Del-1 was also significantly down-regulated.2.Compared with the negative control group(Ctrl),TNF-α induced the chemotaxis of neutrophils to Ha Ca T cells(Ctrl+TNF-α group).Compared with Ctrl+TNF-α group,the chemotaxis of neutrophils to Ha Ca T cells decreased in the over-expressed Del-1 group(OE-Del-1 +TNF-α),while enhanced in the knock-down Del-1 group(sh-Del-1 +TNF-α),suggesting that Del-1 can inhibit the chemotaxis of neutrophils to Ha Ca T cells.3.Compared with the negative control group(Ctrl),TNF-α enhanced the adhesion of neutrophils with Ha Ca T single cell molecular layer(Ctrl+TNF-αgroup).Compared with Ctrl+TNF-α group,the adhesion of neutrophils with Ha Ca T single cell molecular layer decreased in over-expressed Del-1 group(OE-Del-1+TNF-α),while increased in knock-down Del-1 group(sh-Del-1+TNF-α),suggesting that Del-1 can inhibit the adhesion of neutrophils with Ha Ca T cells.4.The results of PCR array screening showed 48 differentially expressed genes in Ctrl+TNF-α group compared with Ctrl group,including 31up-regulated genes and 17 down-regulated genes.Compared with Ctrl+TNF-αgroup,OE-Del-1+TNF-α group had 52 differentially expressed genes,including 30 up-regulated genes and 22 down-regulated genes.There were 50 differentially expressed genes in the sh-Del-1+TNF-α group compared with the Ctrl+TNF-α group,including 42 up-regulated genes and 8 down-regulated genes.The up-regulated genes in Ctrl+TNF-α group,down-regulated genes in OE-Del-1+TNF-α group and up-regulated genes in sh-Del-1+TNF-α group were intersected,and six common differential genes were screened,namely CXCL8(namely IL-8),CXCL1,IL-23 A,IL-1A,IL-5 and C3.CXCL8 with obvious difference multiple and related to neutrophil chemotaxis was selected for RT-q PCR verification.It was confirmed that the level of CXCL8,the most important chemokine of neutrophils,decreased in over-expression-Del-1group and increased in knock-down-Del-1 group.The difference was statistically significant,suggesting that Del-1 can reduce the expression of CXCL8 in keratinocytes.The expression of ICAM-1 was low in normal Ha Ca T cells.In psoriatic cell model,TNF-α up-regulated the expression of ICAM-1,over-expression-Del-1 decreased the level of ICAM-1,while knock-down Del-1 significantly increased the expression of ICAM-1,suggesting that Del-1 can reduce the expression level of ICAM-1 in keratinocytes.5.TNF-α can activate the NF-κB signaling pathway in keratinocytes,resulting in NF-κB-P65 phosphorylation and translocation from cytoplasm to nucleus.The phosphorylation level and nuclear translocation of NF-κB-p65 were decreased in over-expression-Del-1 group,while increased in knock-down-Del-1 group,suggesting that Del-1 can inhibit NF-κB signal pathway.Conclusion1.Del-1 was constitutively expressed in human keratinocytes,and decreased in the epidermis 、 lesion and cell model of psoriasis.The inflammatory factor TNF-α can reduce the expression level of Del-1 in keratinocytes,which may be the underlying mechanism of TNF-α leading to neutrophil infiltration.2.In psoriasis cell model simulated by TNF-α-stimulated Ha Ca T keratinocytes,Del-1 reduced the chemotaxis and adhesion of neutrophils to the epidermis.3.Del-1 may reduce CXCL8 and ICAM-1 expression in keratinocytes by inhibiting NF-κB activation,thereby alleviating neutrophil infiltration in psoriatic epidermis.